Risk stratification method for a patient having a polymorphism

ABSTRACT

A risk stratification method for a patient in a disease state and specifically patients presenting a tumor, includes determining if the patient is a homozygote or heterozygote and further determining the allelic expression for the patient, CC, T/C, or C/T. For patients having the cytosine methylated, they have a C/T allelic expression and patients without a methylated cytosine have a T/C allelic expression, A patient with a TT allelic expression is classified as a highest risk patient, a patient with a T/C allelic expression is classified as a second highest risk, patient, a patient with a C/T allelic expression is classified as a third highest risk patient and a patient with a CC allelic expression is classified as a lowest risk patient. The risk stratification method may further include identification of an abnormal expression or mutation/function of a gene product produced by CTCF binding site 6.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry application ofPCT/US2017/055467, filed on Oct. 6, 2017 and currently pending, whichclaims the benefit of U.S. provisional patent application No. 62/465,730filed on Mar. 1, 2017, and to U.S. patent application Ser. No.15/286,597, filed on Oct. 6, 2016 and currently pending, the entirety ofall application listed above are hereby incorporated by referenceherein.

SEQUENCE LISTING

Sequence listing filename ITP452-Sequencelisting, created on Dec. 20,2016 and having a 1.22 kbyte file size in hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to a risk stratification method for a patientand particularly to a patient having a polymorphism and a tumor, such asa hemangioma and for determination of placenta health, includingprediction of diseases such as an intrauterine growth restriction,pre-eclampsia, eclampsia and placental insufficiency in a patient.

Background

Infantile hemangioma (IH) is the most common tumor of the pediatric agegroup, affecting up to 4% of newborns ranging from inconsequentialblemishes, to highly aggressive tumors. Following well defined growthphases (proliferative, plateau involutional) IH usually regress into afibro-fatty residuum. Despite the high prevalence of IH, little is knownregarding the pathogenesis of disease.

Infantile hemangioma (IH) is the most common tumor of the pediatric agegroup affecting up to 4% of newborns with nearly 60% localized to thehead and neck. These vascular lesions range from inconsequentialblemishes to highly aggressive tumors that can cause eye obstructions orblindness, blockage of airways, facial deformations and ulcerations.During the first year, hemangiomas demonstrate both histology andbehavior that are also commonly noted in malignancy: immature vascularchannels, high mitotic indices, and strong positivity for proliferativemarkers such as Ki-67.

Despite these ominous beginnings, IH remain benign. The growth velocityslowly reverses leading to a “quiescent or plateau” phase of non-growth(1-2 years) and then transitions into a regressive or “involuting” phaseby replacing the once proliferative endothelium with a fibrofattyresiduum (2-10 years). However, these growth phases are a matter ofclinical judgment alone and the exact timing of each varies considerablyamong studies. Despite its prevalence, little is known regarding thepathogenesis of disease. Insulin Like Growth Factor 2 (IGF2) has beenimplicated as an important player in driving the growth of theselesions. IGF2 decreases over six fold from proliferative to involutingIH samples. Furthermore, Beckwith-Wiedmann Syndrome (BWS), wherehemangiomas are considered a supportive finding of the diagnosis, isassociated with duplications or a loss of imprinting of the IGF2/H19locus that leads to IGF2 overproduction. Moreover, explant hemangiomacultures grow strongly in response to exogenous IGF2.

IGF2 is an imprinted gene that is usually only expressed from thepaternal copy. Commonly, DNA methylation of cytosines preceding guanines(CpG's) reinforce DNA imprinting. These so called epigenetic marks inpart determine and are determined by the array of DNA binding proteinscapable of interacting with specific chromatin structures. The endresult of this process is diploid DNA that is potentially identical insequence but chemically, transcriptionally and architecturally distinctin a parent of origin specific manner. This leads to activation of onegiven parental allele and reciprocal silencing of another. The IGF2/H19region of chromosome 11p15,5 serves as a model for the production ofmultiple imprinted transcripts.

There is evidence to support the existence of a common genetic programbetween placental and hemangioma endothelium. The idea of a placentalorigin of hemangioma is evaluated through molecular profiling andgenome-based analysis as described in “Evidence by Molecular ProfilingFor A Placental Origin of Infantile Hemangioma”, Proceedings of theNational Academy of Sciences of the United States, Dec. 19, 2005, CarmenM. Barnes et al., the entirety incorporated by reference herein. Thetranscriptomes of human placenta and infantile hemangioma are shown inthese studies to be sufficiently similar and therefore suggest aplacental origin for this tumor. The unique hemangioma cycle is markedby rapid endothelial cell proliferation, endothelial cell apoptosis, andtumor involution which may mirror the lifetime of a placentalendothelial cells.

SUMMARY OF THE INVENTION

The invention is directed to a risk stratification method for a patientand particularly to a patient having a polymorphism and a tumor, such asa hemangioma and for determination of placenta health, includingprediction of diseases such as an intrauterine growth restriction,pre-eclampsia, eclampsia and placental insufficiency in a patient andprediction of development of postnatal Infantile hemangioma.Identification of the expression of CTCF with parent of origin specificdetermination from the mother and/or from the maternal placenta and/orfrom the fetus or fetal placenta may be used to predict placental healthand stratify patients for diseases such as an intrauterine growthrestriction, pre-eclampsia, eclampsia and placental insufficiency.

The placenta functions as a fetomaternal organ with two components, thefetal placenta, Chorion frondosum, which develops from the sameblastocyst that forms the fetus, and the maternal placenta, Deciduabasalis, which develops from the maternal uterine tissue. A blood samplefrom the mother may be taken for DNA analysis of both the mother and thefetus. In some cases, a simply check swap may be used to provide asample for DNA analysis of the mother. Both of these techniques arenon-invasive. In some cases, a blood sample may be taken from theplacenta or from the fetus for DNA analysis as described herein.

Brother of the regulator of imprinted sites or BORIS, also known astranscriptional regulator transcriptional repressor CTCFL, is a proteinthat in humans is encoded by the CTCFL gene.

With respect to infantile hemangiomas, a reported six fold decrease inIGF2 expression (correlating with transformation of proliferative toinvoluted lesions) prompted a study of the IGF-2 axis further. Asdescribed herein, it has been discovered that IGF2 expression in IH isstrongly related to the expression of a cancer testes and suspectedoncogene BORIS (paralog of CTCF), placing IH in the unique category ofbeing the first known benign BORIS positive tumor. IGF2 expression wasstrongly and positively related to BORIS transcript expression.Furthermore, a stronger association was made when comparing BORIS levelsagainst the expression of CTCF via either a percentage or differencebetween the two, or a rate of increase of % CTCF. A patient with areducing rate of % CTCF may be a higher risk patient, as this mayindicate a more aggressive tumor, as described herein. A common Capolymorphism at CTCF BS6 appeared to modify the correlation betweenCTCF/BORIS and IGF2 expression in a parent of origin specific manner.Moreover, these effects may have phenotypic consequences as tumor growthalso correlates with the genotype at CTCF BS6. This may provide aframework for explaining the clinical variability seen in IH andsuggests new insights regarding CTCF and BORIS related functionality inboth normal and malignant states.

Investigations into the differential regulation and potential role ofIGF2 as it pertains to IH, as well as the expression of CTCF, a knownchromatin insulator element for IGF2 and its antagonist, BORIS (alsoknown as CTCFL for CTCF like) at both the transcript and protein levelsare described herein. The nearby imprinted and maternally expressed H19gene, which shares enhancers with IGF2, was also quantified. Theseresults were then correlated with methylation analysis of key regulatoryregions in the IGF2 and H19 locus. This analysis suggests that a commonpolymorphism within CTCF Binding Site Six, the critical imprintingcontrol region of H19/IGF2, may have both cellular and phenotypicconsequences in a parent of origin specific manner. These findings mayserve as a predictor of clinical behavior of IH and may enable riskstratification for patients having a tumor, and specifically IH.

In an exemplary embodiment, a risk stratification method for a patientin a disease state is accomplished by first determining whether saidpatient is a homozygote patient or a heterozygote patient with respectto CTCF binding site 6. When the patient is a homozygote patient it isthen determined if the patient has a Thymine/Thymine, (TT) allelicexpression or a Cytosine/Cytosine (CC) allelic expression for said CTCFbinding site 6. When the patient is a heterozygote, it is thendetermined whether the cytosine is methylated; whereby when the cytosineis not methylated the patient has a Thymine/Cytosine, (TC) allelicexpression for said CTCF binding site 6; and whereby when the cytosineis methylated the patient has a Cytosine/Thymine (CT) allelic expressionfor said CTCF binding site 6. A patient with a TC allelic expression hasa maternal cytosine and a patient with a CT allelic expression has apaternal cytosine. Risk stratifying can then be determined based on thepatient's allelic expression for CTCF binding site 6, wherein thepatient with a TT allelic expression is classified as a highest riskpatient, the patient with a TC allelic expression is classified as asecond highest risk patient, the patient with a CT allelic expression isclassified as a third highest risk patient and the patient with a CCallelic expression is classified as a lowest risk patient. Furthermore,unique to the disease of vascular tumors in general and infantilehemangioma specifically is the risk of ulceration. A painful conditionwhere the epithelium of the lesion degrades, creating an open woundprone to bleeding. The TT allelic expression is classified at thehighest risk for ulceration.

A highest risk patient having an IH may be prone to a rapidly grow ntumor, ulceration of the tumor, facial deformations, eye obstructions,blockage of airways or any combination of these conditions. A secondhighest risk patient having IH may be prone to moderately to rapidlygrowing tumors, facial deformations, eye obstructions, blockage orairways or any combination of these conditions. A third highest riskpatient having IH may have a slow to moderately growing tumor, facialblemishes. A lowest risk patient having IH may have a slow growingtumor, facial blemishes.

Any suitable method may be used to for determining for a heterozygotepatient whether the cytosine is methylated including, bisulfiteconversion and quantitative methylation sensitive pyrosequencing ordirectly sequencing parental DNA, for example.

A risk stratification method, as described herein, may be initiated fora patient in a diseased state, wherein the patient is in a diseasedstate by identification of a patient tumor. A patient may be consideredto be in a diseased state when the identified tumor is an infantilehemangioma, or any other tumor such as a cancerous tumor including, butnot limited to, breast cancer, ovarian cancer, testicular cancer, livercancer, lung cancer, brain cancer, skin cancer, esophageal cancer,throat cancer, bladder cancer, cervical cancer, colorectal cancer,kidney cancer, leukemia, melanoma, non-Hodgkin lymphoma, ovarian cancer,pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, uterinecancer and any other cancer.

The risk stratification as described herein, may further comprise thestep of identification of an abnormal expression or mutation/function ofa gene regulated by CTCF binding site 6. The abnormal expression ormutation/function of a gene product regulated by CTCF binding site 6 maybe IGF2, H19, H19 antisense, IGF2 antisense, a micro ma within the genelocus, or the gene product may be an isoform.

The risk stratification as described herein, may further comprise thestep of identification of an abnormal expression or mutation/function ofa binding gene product that binds to the CTCF binding site 6. Theabnormal expression or mutation/function of a binding gene product thatbinds to the CTCF binding site 6 may be CTCF, BORIS or a bindingisoform.

The risk stratification as described herein, may further comprise thestep of identification of an abnormal expression or mutation/function ofa binding partner of CTCF or BORIS.

In an exemplary embodiment, a risk stratification method furthercomprising the steps of determining an expression level of BORIS and anexpression level of CTCF with respect to CTCF binding site 6, and thendetermining a percentage of CTCF transcript, such as the percentage ofCTCF with respect to the total of CTCF and BORIS. In an alternativeembodiment, a difference in the amount of CTCF to the amount of BORISmay be used. A risk stratification method may take into account theallelic expression, as described herein, and may further take intoaccount the % CTCF, wherein, when the % CTCF is less than 20%, thepatient is classified as a highest % CTCF risk patient. wherein when the% CTCF is less than 50% but greater than 20%, the patient is classifiedas a second highest % CTCF risk patient, wherein when the % CTCF is lessthan 80% but greater than 50% the patient is classified as a thirdhighest % CTCF risk patient and wherein when the % CTCF ratio is lessthan 100% or less and greater than 80% the patient is classified as alowest % CTCF risk patient. Furthermore, the risk stratification methodmay include the rate of reduction of % CTCF, wherein a rapid drop in %CTCF may indicated a higher risk of an aggressive tumor, or a tumor thatgrows quickly.

In an exemplary embodiment, a risk stratification method furthercomprising the steps of determining an expression level of BORIS and anexpression level of CTCF with respect to CTCF binding site 6, and thendetermining a CTCF-BORIS difference which is a difference in theexpression level of CTCF to the expression level of BORIS. The patientmay then be risk stratified according to said CTCF-BORIS differencewherein, when the CTCF-BORIS difference is less than zero, the patientis classified as a highest CTCF-BORIS risk patient; and when theCTCF-BORIS difference is greater than zero, the patient is classified asa lowest CTCF-BORIS risk patient.

In an exemplary embodiment, the risk stratification method as describedherein, may be used to predict a response of medication to the tumor.For example, a patient with a TT allelic expression and classified as ahighest risk patient may have a predicted medication response that ismost effective, a patient with a TC allelic expression and classified asa second highest risk patient may have a predicted medication responsethat is second most effective, a patient with a CT allelic expressionand classified as a third highest risk patient may have a predictedmedication response that is third most effective and a patient with a CCallelic expression and classified as a lowest risk patient may have apredicted medication response that is least effective. Patientsclassified as the highest risk patients may have the most effectiveresponse to medication whereas patients classified as the lowest riskpatients may have the least effective response to medication. Medicationmay be beta blockers, cortical steroids, alpha interferon, and/or IGF2receptor blockers. Preferred medications are beta blockers and corticalsteroid. A most effective response or highly effective response tomedication includes slowing of the growth of the tumor, reduction insize of the tumor without rebound or relapse, closure of ulceration orhealing or ulceration. A second most effective response to medicationincludes reduction in size of the tumor without rebound or relapse. Tthird most effective response to medication includes reduction in sizeof the tumor without rebound or relapse. A least effective response tomedication includes reduction in size of the tumor, or no reduction insize of the tumor.

The medication response prediction method may further comprise the stepof determining the disease state of said patient by identification of anabnormal expression or mutation/function of a gene product produced byCTCF binding site 6. The abnormal expression or mutation/function of agene product regulated by CTCF binding site 6 may be IGF2, H19, H19antisense, IGF2 antisense, a micro ma within the gene locus, or the geneproduct may be an isoform.

The method of determining this polymorphism and allelic expression ofthe gene product regulated by CTCF binding site 6 for the fetus or foran infant, may also be used to predict placental health and riskstratify mothers for placental diseases including an intrauterine growthrestriction, pre eclampsia, eclampsia, placental insufficiency. DNAanalysis of the mother and/or the fetus may be used in this predictivemodel. As with the risk stratification model described herein, apredicted model may be based on the allelic expression for CTCF bindingsite 6 of the mother, the fetus or some combination thereof. A motherwith a TT allelic expression may be classified as a highest stratifiedrisk patient, a mother with a TC allelic expression may be classified asa second highest risk patient, a mother with a CT allelic expression maybe classified as a third highest risk patient and a mother with a CCallelic expression may be classified as a lowest risk patient. Thevarious combinations of allelic expression of the mother and fetus mayfurther be used in a risk stratification method. When the mother and thefetus both have a TT allelic expression, this may correlate with thehighest risk patient for placental health issues, the mother and fetushave a CT or TC allelic expression, this may correlate with a secondhighest risk patient for placental health issues and when both themother and fetus have a CC allelic expression, this may correlate withthe lowest risk patient for placental health issues. It is to be notedthat data to determine correlations between the allelic expression ofCTCF binding site 6 of the mother and/or the fetus may alter the riskstratification categories as provided herein.

A G/A polymorphism approximately 130 base pairs downstream of rs10732516was found to be in strong linkage disequilibrium with the (C/T)polymorphism at CTCF BS6 This polymorphism is rs2107425. Thispolymorphism was detected by using differential primer binding, seeincorporated reference Tost et al, for details. The A polymorphism atrs2107425 was strongly associated with the T polymorphism at CTCF BS 6(rs10732516) in the samples. Thus, it is conceivable that nearbypolymorphisms within the CTCF BS6 locus would be used as a surrogate fora test at rs10732516. This concern is underscored by the fact that largeareas of linkage disequilibrium of over 500 base pairs have beendocumented. Shoemaker et al¹. This has included regions in IGF2,Shoemaker et al. Given the high level of linkage disequilibrium weobserved at CTCF binding six between re10732516 and rs2107425 we proposethat the sequence between CTCF binding site 5 and CTCF binding siteseven (this includes CTCF binding site six and rs10732516) be consideredone quantitative trait locus. Therefore, for the purposes of thisinvention, a polymorphism as described herein, may be determined byanalysis of any sequence between CTCF binding site 5 and CTCF bindingsite 7. The placental health predictive indicators as well as the riskstratification methods can be based on analysis of any sequence betweenCTCF binding site 5 and CTCF binding site 7.

Allele-specific methylation is prevalent and is contributed by CpG-SNPsin the human genome. The risk stratification method, as describedherein, may be characterized from CTCF binding site 6, or a rangeupstream and downstream, because of high linking disequilibrium, asdescribed in Robert Shoemaker, Jie Deng, Wei Wang and Kun Zhang GenomeResearch 2010 July; 20(7): 883-889, the entirety of which is herebyincorporated by reference. The risk stratification method of the presentinvention may be performed on base pairs up to 500 base pairs from CTCFbinding site 6, or up to about 350 base pairs from CTCF binding site 6,or from about 150 base pairs from CTCF binding site 6. or from CTCFbinding site 5 to CTCF binding site 7.

The summary of the invention is provided as a general introduction tosome of the embodiments of the invention, and is not intended to belimiting. Additional example embodiments including variations andalternative configurations of the invention are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate embodiments of the invention, andtogether with the description serve to explain the principles of theinvention.

FIG. 1 shows a drawing of an infantile hemangioma.

FIG. 2A shows a diagram representing the genotyping via directsequencing of blood samples.

FIG. 2B shows a chart of bisulfite conversion and quantitativemethylation sensitive pyrosequencing.

FIGS. 3A and 3B show a chart of IGF2 transcription by clinical stagewherein FIG. 3A shows IGF2 Transcript and FIG. 3B shows % CTCF.

FIG. 4A shows a graph of the percent CTCF with respect to CTCF and BORISversus the age of the lesion.

FIG. 4B shows the CSUM of % CTCF versus the age of the lesion.

FIG. 5 shows a graph of IGF2 Transcript versus the % CTCF.

FIG. 6 shows Western Analysis of 24 IH Samples Via 5 Independent WesternBlots.

FIG. 7 shows a schematic integrating CTCF and BORIS expression via bothtranscript and protein with clinical stage.

FIGS. 8A and 8B show a graph of the parent of origin, paternal andmaternal respectively, specific effects of CTCF BS6 on IGF2 and H19transcription.

FIG. 9A shows a graphs of percent methylation versus CpG number.

FIG. 9B shows a graph of percent methylation of the H19 promoter bypyrosequencing versus CTCF transcript.

FIG. 9C shows methylation of the H19 promoter.

FIG. 9D shows a sequence showing the H19 promoter, CpG#4 of thebisulfite sequencing test corresponds to the CCCGGG Pst1 digestion siteof the Southern analysis.

FIG. 10A shows a graph of the diameter of an infantile hemangioma versusthe age of the lesion in days for TT and non-TT patients.

FIG. 10B shows a graph of the diameter of an infantile hemangioma versusthe age of the lesion in days for TC, C/T and C/C patients.

FIGS. 11A to 11D show portions of Master Table.

FIG. 12 is a table of the summary of clinical data. Retrospectivelycollected results with associated descriptive information.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Corresponding reference characters indicate corresponding partsthroughout the several views of the figures. The figures represent anillustration of some of the embodiments of the present invention and arenot to be construed as limiting the scope of the invention in anymanner. Further, the figures are not necessarily to scale, some featuresmay be exaggerated to show details of particular components. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting, but merely as a representative basis forteaching one skilled in the art to variously employ the presentinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such process, method,article, or apparatus. Also, use of “a” or “an” are employed to describeelements and components described herein. This is done merely forconvenience and to give a general sense of the scope of the invention.This description should be read to include one or at least one and thesingular also includes the plural unless it is obvious that it is meantotherwise.

Certain exemplary embodiments of the present invention are describedherein and are illustrated in the accompanying figures. The embodimentsdescribed are only for purposes of illustrating the present inventionand should not be interpreted as limiting the scope of the invention.Other embodiments of the invention, and certain modifications,combinations and improvements of the described embodiments, will occurto those skilled in the art and all such alternate embodiments,combinations, modifications and improvements are within the scope of thepresent invention.

This application incorporates by reference in its entirety, thefollowing publication:

-   -   Brent Schultz, Xiaopan Yao, Yanhong Deng, Milton Waner,        Christopher Spock, Laura Tom, John Persing, Deepak        Narayan, (2015) A Common Polymorphism Within The IGF2 Imprinting        Control Region Is Associated with Parent of Origin Specific        Effects in Infantile Hemangiomas, PLOS ONE| DOI:10.1371/journal.        pone.0113168

Materials and Methods

This article describes a study that involved analyzing post excisionaltissue from surgical candidates. The decision to operate was in no wayinfluenced by the study. Clinical data was gathered retrospectively fromthis same group. All surgical candidates had clinical measurementsavailable for analysis. All samples and clinical data were collected inaccordance with the approved HIC protocol (#0507000430) as reviewed bythe Yale University Medical School IRB. This protocol was approvedspecifically for this study. Written consent was obtained from eachpatient's legal guardian prior to surgery. All data obtained includingclinical measurements were stored in a de-identified format.

Specimen Collection:

Please refer to the Master Data Table for details, as shown in FIGS. 11Ato 11D. Those specimens later confirmed to be hemangioma tissue, viaGlut-1 positive histology, were considered for this project. Further,only discrete solitary lesions that were not found in the setting of asyndrome were considered. Those patients where prior surgical resectionsof the lesion were performed were also deemed ineligible. Of note, thoselesions previously treated with laser were not excluded, as the effectsof laser treatment are relatively superficial. However, during specimencollection, all areas that appeared grossly to be affected by lasertreatment were excised before further processing. Briefly, forty-twosamples were collected (See FIGS. 11A to 11D for details.) Of these, twosamples (#41 and #42), were excluded from all analyses; sample #41 wasGlut-1 negative on histology and #42 was subject to prior resections.Nineteen IH samples were selected at random for methylation analysis ofthe H19 promoter by southern. These 19 samples were also analyzed formethylation specific pyrosequencing of the same region. An additionaltwo samples (numbers 1 and 14) were also subjected to H19 methylationspecific pyrosequencing to bolster the number of samples with both H19methylation and transcriptional data. Regarding transcriptionalanalysis, nineteen samples were found to have suitably intact RNA forquantitative RT PCR. Specimens for transcriptional analysis wereseparated into three categories: 1) Proliferative, 2) Quiescent, and 3)Involuting phases. As a lesion's stage is, by definition, clinical, anexperienced physician staged the IH at the time of surgery. Dataregarding clinical stage was gathered prospectively. The determinationof the clinical stage was made by one of three highly experiencedsurgeons regarding vascular anomalies, using interval growth, patientage and the color/turgor of the lesion at the time of resection ascriteria. General characteristics of these categories are as follows: 1)proliferative hemangiomas were generally less than 1.5 years of age withinterval growth between the last two clinical visits preceding surgery,no lightening of lesion color was noted. 2) Quiescent hemangiomas: nointerval growth between the last two clinic visits preceding surgery,lightening of color also played a factor in these determinations.Involuting hemangiomas: interval regression by measurement between thelast two clinic visits preceding surgery, further color changes wereoften but not always noted.

Master Data Table. As shown in FIGS. 11A to 11D, all samples areassigned arbitrary numbers for ease of reference. Samples arecategorized according to which set of experiments were performed, thenby paternal/maternal genotype regarding the IGF2 rtPCR experiment Allsub categories are then sorted by age at resection. All quantitativedata is collated with clinical descriptors. Please see methods sectionunder specimen collection for details regarding the selection ofindividual samples for each experiment. Master Table is provided in fourexpanded portions in FIGS. 11A to 11D.

In total 34 samples were genotyped for a polymorphism within CTCF BS6and parental contributions were determined for heterozygotes (see FIG. 2and bisulfite sequencing methods for details.) Only lesions of the headand neck were included. Of these samples, 3 were excluded because theywere not on the head or neck. Two other samples were excluded becauseone was not Glut-1 positive on histology and one had a previousresection of the same lesion prior to evaluation. Thus 29 individualswere included in this analysis. Charts were retrospectively reviewedfrom patients treated either at the Yale University Plastic SurgeryCenter (New Haven Conn.), or the Vascular Birthmarks Institute (NewYork, N.Y.). The age of the lesion was then compared to the size of thelesion as determined below. These data were plotted and separated byCTCF BS6 genotype and parental contribution in heterozygotes. ANCOVAanalyses were then performed on putative growth curves. The age at thetime of resection, with corresponding size, was used only forproliferating lesions. For those lesions that were resected at the timeof involution, or quiescence, the size of the lesion at the clinicalvisit where quiescence was first noted was used. In the case of medicalinterventions, the size of the lesion before a response was noted wasused. This information was used to create a clinical table of resultswere factors such as ulceration, steroid/chemotherapeutic, and lasertreatment were also noted (note that no beta blockers were used in thesample population.) Thus, different ages are associated with mostindividuals when comparing the clinical data table and the master datatable. For instance: Sample #7 has an age of 2304 days assigned in theMaster Data Table. However, in the Clinical Table the age assigned tosample #7 is 1050 days. The difference in age assignments represent theage of the patient when the lesion was excised (this age was used forthe molecular analyses) versus the age of the patient either before thefirst response to medical intervention was noted or when the lesionfirst entered the quiescent phase. Thus, for sample number 7: The lesionentered the quiescent phase (as determined retrospectively) at age 1050days but was then excised at age 2304 days. Regarding the assessment oflesion size, if multiple dimensions were given, the largest was used. Insome cases, only one dimension was given so volumetric estimations couldnot be calculated for every patient. Thus, patients' lesions werestandardized to a greatest diameter equivalent. This measurement wascorrelated with clinical photographs when available. All data utilizedvaried by less than 10% between stated measurement and photographicestimation when available. Lesions were classified into one of threegrowth phases: proliferative, quiescent and involuting. Only sizes oflesions that were in the proliferative or quiescent phase were used inthis study. All data was stored in a de-identified format with a uniqueaccession number for each patient.

DDNA Preservation and Extraction

Immediately following tissue resection, DNA was isolated using theQiagen DNeasy Tissue Mini Kit according to the manufacturer's protocol.Only samples with an A260/A280 measurement of 1.8 or above that ran as asingle band on the gel were further analyzed.

FIG. 2. Deducing Parental Contributions From Direct Sequencing andBisulfite Pyrosequencing. FIG. 2A: 29 patients were genotyped via directsequencing of blood samples for a known polymorphism within the coreCTCF BS6 sequence (rs10732516.) All homozygous genotypes could bededuced from this information alone. FIG. 2B: All samples (heterozygotesand homozygotes) were subjected to bisulfite conversion and quantitativemethylation sensitive pyrosequencing. Methylation occurs only on thepaternal chromosome for CTCF BS6. In normal tissue, such as patientmatched control blood, this assay is capable of isolating the genotypeof the paternal chromosome. As thymidine cannot be methylated, thoseindividuals with a paternal T at rs10732516 were not methylated atCpG#5. Paternal C carrying individuals were methylated at CpG#5. Thus,the maternal and paternal contribution to CTCFBS6 can be deduced. Thisassay sidesteps the need for directly sequencing parents' DNA andeliminates the potential ambiguity ensuing from heterozygous parents.Note: The methylation values of this assay are subject to primer bias,Tost et al (25.) This is evident by the 3 distinct groupings ofmethylation levels, which are artifactual.

RNA Preservation and Extraction

Immediately following tissue resection, 100-500 mg of tissue was storedin Quiagen RNA Later solution according to the manufacturer's protocol.RNA was extracted via liquid nitrogen powder homogenization usingInvitrogen Trizol reagent according to the standard protocol. 10 μg oftotal RNA from each sample was then treated with DNase Qiagen mini-elutecolumns according to manufacturers specifications. RNA integrity wasthen assessed using Agilent bioanalyzer 2100 (provided as a service ofthe Keck Center at Yale University.) Those samples with 18s/28s ratio of1.8 or greater were converted into cDNA using the ABI 4368813 cDNAarchive kit. All samples were then stored at −80 degrees C.

Quantitative rtPCR for CTCF, BORIS, H19 and IGF2

Nineteen IH samples with suitable RNA, as previously specified, weresubjected to fluorescent quantitative RT-PCR using ABI Taqman primersthat were previously validated by the manufacturer and spanned intronexon boundaries. For reasons of sample scarcity, not all samples weresubjected to each assay (See FIG. 1 for details.) The assays were:IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1,CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.Gene quantification was performed using the standard curve method viapooled sample cDNA (equal contributions from each sample) and successivetwo fold dilutions, beginning from 50 ng and ending with 0.39 ng. Allreactions were performed on the ABI 79005 thermocycler using defaultcycling conditions previously optimized for these assays. Reactions wereperformed in duplicate and average CT values, if they agreed within 0.4cycles, were used to calculate absolute quantity. Three runs of RT PCRwere performed with overlapping samples in each run to allownormalization of the data. Not all samples were subjected to every assaydepending upon sample quantity. Of Note: Sample #4 does not have an H19transcription value, as on duplicate plating for rtPCR, the CT valuesdid not agree within 0.4 cycles. Furthermore, samples 15-19 were thefinal rtPCR of the three runs performed and due to sample scarcity andthe need to construct standard curves from pooled samples, only CTCF andBORIS rtPCR's were performed.

Western Analysis

24 samples were subjected to Western analysis. As this process is tissueintensive, younger samples such as #21 and #23 could only be used forthis analysis as insufficient tissue was left for further processing.Other samples were selected biased toward analyzing those samples withtranscriptional data in order to compare transcriptional phenomena totranslational. However, as the analysis proceeded, presentation gelswere constructed to demonstrate key transition points in CTCF and BORIStranslation in samples that had not been treated with steroids. Briefly,50 mg of each sample were processed with a rotary homogenizer in 200 mlof RIPA lysis buffer. After centrifugation lysates were created using astandard beta-mercapto-ethanol with SDS. PAGE was performed with 36 μgof protein per well in NuPage 10% Bis-Tris precast gels inMOPS buffer at100 volts. PAGE separated proteins were then transferred for two hoursto a PVDF membrane (Bio-Rad) in a standard transfer buffer at 100 mAmps.Anti BORIS antibody (Abcam 18337) was used at 1/5000 dilution in TBSTwith 5% cows milk overnight. Two concentrations of anti-CTCF wereused—1:10,000 and 1:5,000—to better visualize late and early rises inCTCF protein (see FIG. 3 legend for details) As anti-CTCF and anti-BORISwere both rabbit polyclonal antibodies they could be visualizedsimultaneously on the same film following incubation with theanti-rabbit secondary conjugated to horseradish peroxidase and ECLtreatment. Images were then scanned and adjusted for brightness andcontrast in Adobe Photoshop.

Bisulfite Methylation Analysis Using Quantitative Pyrosequencing

This method was first described by Grunau et al and Dupont et al.Protocols specific for each assay in this study are available in S1Supplementary Information or upon request. Incomplete bisulfiteconversion was detected by designing amplicons that contained at least 1unmethylated cytosine. Primer bias was controlled for by establishingmethylation curves of 100% methylated DNA titrated against known amountsof whole genome amplified PCR products that, by definition, areunmethylated. These methylation curves allow experimental samples to becalibrated against known standards. Primers for Exon 9, the H19 promoterand CTCFBS6 as well as the bisulfite-converted sequences they amplifyare available in S1 Supplementary Information. The presence of an A/Gpolymorphism, approximately 130 base pairs downstream of CTCF BS6 leadsto primer bias and distorts the absolute methylation values of CTCF BS6.Tost et al.

Deducing Parental Contributions of Alleles at CTCF BS6

All samples were subjected to direct sequencing of CTCF BS6 containingthe polymorphism rs10732516. Primer design and reaction conditions areavailable in S1 Supplementary Information. The DNA samples weresubjected in parallel to methylation sensitive pyrosequencing of thesame polymorphism; please see the section titled “Specimen Collection”for further details. Comparing these results allows each parentalcontribution to be deduced, see FIG. 1 for full details.

As shown in FIG. 3, IGF2 transcription by clinical stage shows aninverse relationship to the % CTCF of identical stage. FIG. 3A: IGF2mRNA levels were approximately 6× lower in the involuting samplescompared to their proliferating counterparts. Proliferating vs.involuted p=0.02. Proliferating vs. quiescent p=0.0081, Quiescent vsinvoluting p=0.01. Wilcoxian Rank Sum Test. Error bars representstandard deviation. FIG. 3B: % CTCF changes significantly according toclinical stage. Profit vs. quiescent p=0.1, quiescent vs. invol p=0.06,prolif vs. invol p=0.63, Wilcoxian two sample test Error bars representstandard deviation. Note: All samples in the IGF2 analysis were includedin the % CTCF analysis, with additional samples.

Genomic Southern Analysis for the H19 Promoter

Eighteen were analyzed at a CLIA certified molecular diagnosticslaboratory where this assay is performed as a clinical test forBeckwith-Wiedemann Syndrome. The assay is originally described by Debaunet al. Norms for this test were previously established with 30 normalcontrols at 55% methylation with a standard deviation of 5%. All sampleswere run with a normal and Beckwith-Wiedemann control. The assayexploits a CCCGGG site in the H19 promoter that is cut by themethylation sensitive restriction enzyme Pst1.

Statistical Analysis

Descriptive statistics were used to present patient characteristics. Thedifference in expression of IGF2 transcript across the threedevelopmental stages of IH was evaluated using a Kruskal-Wallis test. Toevaluate if the relative amount of CTCF compared to BORIS transcriptchanges predictably over time, change point analysis was performed.Change point analysis indicates the likelihood that a change intranscript expression occurred in the sample population by confidencelevel and a confidence interval regarding when those changes occur. The% CTCF [CTCF/(CTCF+BORIS)×100] was used to develop a change point modelthat was then compared against clinical staging and IGF2 expression inthe sample population. A full explanation of the methods used, as wellas a shareware change-point analyzer is presented as an online resource:Taylor, Wayne A, (2000), “Change-Point Analysis: A Powerful New Tool ForDetecting Changes,”(http://www.variation.com/cpa/tech/changepoint.html.) To evaluate theassociation of IGF2 transcript and the relative amounts of CTCF, alinear regression model was fitted, with the % CTCF and age ascovariates. The correlation and partial correlation were alsocalculated. Partial correlations indicate what percentage of variance inIGF2 can be explained by CTCF % alone. Analysis of the covariance model(ANCOVA) was fitted to examine if the correlation between the IGF2transcript and the difference between CTCF and BORIS varied by thepaternal genotype at CTCF BS6, once adjusted by age.

Results Master Data Table

In total, 40 samples were analyzed on a molecular basis. A descriptionof basic demographics, with genotypes at CTCF BS 6 and transcriptexpression values for IGF2 H19, CTCF and BORIS with correlativemethylation data was compiled. Please see FIG. 11A to 11D: Master DataTable for details. This table can be utilized to confirm any statisticalanalysis presented in this study.

Expression of IGF2, CTCF and BORIS

FIGS. 4A and 4B Analyzing the Percentage of TCF Compared to Total CTCFand BORIS Transcript.

FIG. 4A Using the two samples with the lowest % CTCF (420 and 418 daysas common points (Purple)) two curves with high correlation to age canbe appreciated. % CTCF steadily decreases as lesions age untilapproximately 400 days (red and purple points), then CTCF once againincreases compared to BORIS (purple and blue points). This roughlycorrelates with the transition from proliferative to quiescent lesions.FIG. 4B CSUM of % CTCF demonstrates statistically significant variationabout the mean of % CTCF according to age. For a full explanation of theCSUM data and commensurate change point analysis.

IGF2 transcription differed significantly by clinical stages (p<0.0001,Kruskal-Wallis test). Plateau stage lesions expressed significantlyhigher levels of IGF2 than proliferating (p=0.0081, Wilcoxon rank sumtest) and involuted samples (p=0.02). Involuted hemangiomas expressedthe lowest levels of IGF2, approximately 6× lower than theirproliferating counterparts (p=0.01),

To potentially explain the changes in IGF2 transcription, quantitativeRT-PCR was performed for CTCF and BORIS. CTCF and BORIS are co-expressedin all samples. However, the percentage of CTCF transcript compared tototal CTCF and BORIS in a given sample [CTCF/(CTCF+BORIS)×100] variedsignificantly over developmental time (FIGS. 3B and 4A). This wasconfirmed by a change point model (FIG. 4B): the Y axis is thecumulative sum (CUSUM) of the differences between % CTCF and the averagevalue of % CTCF. A segment of the CUSUM chart with an upward slopeindicates a period where the values tend to be above the overallaverage. Likewise, a segment with a downward slope indicates a period oftime where the values tend to be below the overall average. Based onthis analysis, two change points, one estimated at 418 days and theother at 1277 days, were detected. Prior to approximately 418 days (90%CI: 368-547 days), the value of % CTCF tends to maintain a higher levelwith an average value in this stage equal to 59%. In the second stage(418-1277 days), the level of % CTCF is low with an average 28%. Afterthe second change point at approximately 1277 days (90% Cl: 760-1500days). % CTCF has recovered to a high level again with the average 66%.See S2 Supplementary Information for a bar graph analysis of the changepoint model of % CTCF expression. This result is highly similar to theresults obtained by separating samples according to clinical stage (S3Supplementary Information) % CTCF transcription in IH varies accordingto clinical stage (FIG. 3B) Furthermore, the % CTCF varied inverselywith IGF2 transcription (compare FIGS. 3A and 3B) These two graphsclearly demonstrate that a higher % CTCF corresponds with lower levelsof IGF2 expression. Moreover, a strong positive correlation was detectedbetween BORIS and IGF2 transcription (p=0.0028). Though CTCF alone doesnot significantly correlate with IGF2 transcript levels, taking bothCTCF and BORIS into account using % CTCF is the strongest predictor ofIGF2 mRNA expression p=0.0004(FIG. 5).

As described herein, a reduction, especially a rapid reduction, in the %CTCF, as expressed in FIGS. 4A, 4B, and 5, may be an indication of anaggressive tumor and may be used to risk stratify the patient. A % CTCFmay be determined as a proportion of the sum of the expression level ofCTCF and expression level of BORIS. It has been found that a reductionin the % CTCF overtime, or rate of % CTCF, in indicative of a moreaggressive tumor, thereby putting the patient in an higher riskclassification. For example, as shown in FIG. 4A, if the line fit of %CTCF versus age in days has a negative slope, then a patient may beclassified in as a highest risk patient and if the slope is positive,then the patient may be classified as a lowest risk patient.

As shown in FIG. 5, IGF2 transcript levels correlate inversely with thepercentage of CTCF compared to a total of CTCF+BORIS. This datarepresents the first demonstration of the potentially antagonisticeffects of CTCF and BORIS on a target gene's transcription through acontinuous curve. P=0.004 ANCOVA Model. Age effect was not significantin the model p=0.241. The % CTCF is a stronger statistical predictor ofIGF2 expression than BORIS alone 0.0004 vs 0.0028 respectively. (N=15)

FIG. 6. shows Western Analysis of 24 IH samples via 5 independentWestern Blots. Twenty samples with 4 blots depicted, demonstrates 4stages of CTCF and BORIS expression. 6-1: A low concentration ofanti-CTCF (1:10,000) demonstrates the complete spectrum of CTCFexpression with increases early (210 to 418) and late (760 to 987) inprotein expression. (Note, a testes negative control was included aswell as a venous malformation denoted as “VM.”) 6-2 through 6-4 wereprobed with 1:5000 concentration of anti-CTCF that more clearlydemonstrates the early rise in CTCF that occurs after 367 days. 6-2suggests an early increase in BORIS with precipitous downregulationafter 244 days. 6-3 and 6-4 expand this critical age range demonstratinga period from 244 days to 367 where BORIS is downregulated but CTCF isnot yet upregulated. Note, samples marked with an asterisk were alsosubjected to CTCF and BORIS rtPCR.

Western analysis of CTCF and BORIS confirms and expands upon thetranscript data (FIG. 6) As expected in proliferating lesions, BORIStranscript and protein levels steadily rise in early stage samples (FIG.4A transcript data, FIG. 6-2 Western Analysis.) Furthermore, during thetransition from quiescent to involuting samples, CTCF mRNA and proteinincrease compared to BORIS (FIGS. 3B, 4A and 4B transcript, and FIG. 6-1protein.) Thus, the western and transcriptional data globally confirmone another at the endpoints of IH development. However, the proteindata suggests a third change in CTCF and BORIS levels that thetranscript change point analysis does not. This third proteomic changeappears to take place at the late proliferating to early quiescentphase. It coincides with the so-called late proliferative stage in IHthat is suggested by clinicians but not universally accepted. These dataprovide the first molecular support for what was previously a clinicalcategory: the late proliferative stage of IH growth. The relativeexpression of CTCF and BORIS via both transcript and protein levels, ispredictive of clinical stage and IGF2 expression. See FIG. 7 fordetails.

FIG. 7. shows a schematic integrating CTCF and BORIS expression via bothtranscript and protein with clinical stage. The Western analysissuggests 4 stages of CTCF and BORIS (see FIG. 3, panels 1 to 4) eachstage leading to higher levels of CTCF expression relative to BORIS.These interval changes in protein expression closely correlate withclinical stage. Furthermore, bars above the stages represent the 95%confidence intervals of the two change points identified by quantitativertPCR. These data show remarkable agreement reinforcing the idea thatrelative CTCF and BORIS expression levels closely mirror the clinicalstage of the lesions tested. Of note, the CTCF and BORIS protein dataalso suggest a molecular basis for a late proliferative stage.

The CTCF to BORIS transcript difference (C-B) predicts IGF2transcription according to the paternal allele at CTCF BS6. This studyutilizes existing technologies: Direct sequencing of the knownpolymorphism of CTCF BS6 (rs10732516) with a previously describedmethylation assay for CTCF BS6. Applying these two assays in a novelmanner (see FIG. 2) allowed us to deduce both maternal and paternalcontributions to CTCF BS6—which will be referred to as the maternal andpaternal contribution (FIG. 8.) This paternal contribution likely hassignificant effects on IGF2 production as it relates to CTCF and BORIS.

IGF2 mRNA is demonstrated to be inversely related to CTCF and positivelycorrelated to BORIS transcripts when plotted against % CTCF (FIG. 5).

By using the difference between CTCF and BORIS (C-B) rather than the %CTCF, this relationship can be differentiated by the paternallycontributed allele at CTCF BS6 (FIG. 8A). The paternal allele of acommon C/T polymorphism within CTCF BS6 (rs10732516) corresponds withtwo strikingly different CTCF-BORIS vs. IGF2 curves. After ageadjustment, the effect of CTCF-BORIS on IGF2 transcription wassignificantly different between patients bearing different paternalalleles (p=0.05, ANCOVA model) and there is a strong correlation betweenIGF2 expression and CTCF-BORIS (p=0.0007). The samples bearing apaternal C allele, appear to demonstrate a six fold steeper slope ofIGF2 mRNA relative to the CTCF−BORIS difference, compared to theirpaternal T bearing counterparts. This allele was identified in bothtissue and patient matched control blood. Of note, heterozygote analysisrevealed no clear relationship regarding these factors according to thematernal allele (p=0.95, ANCOVA model). It remains a possibility thatthe paternal allele effect may be steroid treatment driven as moresamples with the paternal T allele were treated with steroids than thepaternal C allele. This potential bias was investigated with an oddsratio calculation sorting steroid treatment according to paternalgenotype. The odds ratio suggested that paternal T samples were morelikely to be treated with steroids but this result did not reachstatistical significance. As the allele specific analysis was done ononly proliferative samples the odds ratio calculation was performedtwice, once including involuted samples and once to their exclusion.However, it is acknowledged that the odds ratio suggested a potentialsteroid treatment bias in paternal T samples, which may have becomesignificant in a larger patient cohort. Lastly, IGF2 expression in IHwas mono-allelic in all 5 informative heterozygotes tested for a knownIGF2 polymorphism in exon 9. IGF2 imprinting status appears to bemaintained despite BORIS expression.

H19 transcript levels correlate positively with CTCF mRNA according tothe maternally contributed allele at CTCFBS6. After age adjustment, CTCFtranscript levels alone correlated positively with H19 transcription butonly when separated by maternal genotype ((p=0.0150, FIG. 8B) Moreover,this positive correlation is significantly different among patients withdifferent maternal alleles (p=0.0129, ANCOVA). The correlation betweenCTCF and H19 transcription is stronger in patients bearing a maternal Callele compared to their maternal T counterparts. There were noidentifiable relationships between H19 transcription and either thepaternal genotype at CTCF BS6 or BORIS mRNA, p=0.8 ANCOVA (S4ASupplementary Information.) There also appeared to be no relationshipbetween H19 expression and clinical stage of the hemangioma (S4CSupplementary Information).

It remains a possibility that the maternal allele effect may be steroidtreatment driven as more samples with the maternal T allele were treatedwith steroids than the maternal C allele. This potential bias wasinvestigated with an odds ratio calculation sorting steroid treatmentaccording to maternal genotype. The odds ratio suggested that maternal Tsamples were more likely to be treated with steroids but this result didnot reach statistical significance (See S4F Supplementary Information.)However, it is acknowledged that this odds ratio may have becomestatistically in a larger sample size; the effect was not great enoughto significantly bias the sample size.

CTCF transcript levels alone correlate with demethylation of the H19Promoter. All IH samples tested demonstrated significant hypomethylationat the H19 promoter compared to matched patient blood controls (FIG.9A). This was demonstrated with bisulfate specific pyrosequencing (FIG.9B) and confirmed with methylation sensitive enzyme digest and southernanalysis (FIG. 9C). As pyrosequencing is quantitative, these data werecorrelated with CTCF and BORIS expression in matched samples. Decreasedpromoter methylation correlated with higher levels of CTCF (p=0.015;simple regression. FIG. 9B). Yet, this finding may be subject toconfounders as it is not statistically significant after age adjustment(p=0.17). As the H19 promoter is paternally methylated and maternallydemethylated, a methylation level below 50% would entail demethylationof the paternal allele in at least a subset of cells within a givensample. However, no bi-allelic expression of H19 could be detected in 5heterozygote samples (S5 Supplementary Information.) Furthermore, H19promoter demethylation did not strongly correlate with H19 expression(S6 Supplementary Information.)

FIG. 9. shows CTCF expression and H19 promoter methylation. FIG. 9A:Increased CTCF transcript level correlates with demethylation of the H19Promoter. Those samples with the highest CTCF expression were the leastmethylated ranging from 34% to 14%. However, demethylation of the H19promoter did not correlate strictly with H19 transcript expression (S6Supplementary Information). FIGS. 9B and 9C—The H19 promoter (see FIG.9D) is hypomethylated, demonstrated by bisultife convertedpyrosequencing (9B) and methylation sensitive restriction digest withsouthern hybridization (9C.) 25 IH samples, and 13 matched bloodcontrols were subjected to bisulfite converted pyrosequencing. 13 IHsamples and 13 matched blood controls were subjected to southernanalysis with methylation sensitive Pst1 digestion. Two representativegels show, 5 IH samples, a Beckwith-Weidman positive control and a 50%methylated normal control. FIG. 9D: sequence showing the H19promoter—CpG#4 of the bisulfite sequencing test corresponds to theCCCGGG Pst1 digestion site of the Southern analysis. Other CpG's testedare in bold. This CpG is in close proximity to the transcription startsite of H19 (blue arrow) and an overlapping putative CTCF binding siteidentified by positional weight matrix analysis.

Multiple imprinted sites within the IGF2/H19 locus are abnormallymethylated in IH compared to matched control blood.

It remains a formal possibility that the normalization of CTCF to BORISratios, as well as decreased IGF2 transcription, in involuting andinvoluted samples is not due to an intracellular phenomenon but ratherthe incremental replacement of abnormal IH tissue (vascular stroma) withnormal tissue (fat.) Thus, the results presented are the product oftissue heterogeneity. It is acknowledged that IH lesions transform froma vascular tumor into a fibrofatty residuum; therefore, the transitionalphases are by definition composed of heterogeneous cell populations.However, no evidence was observed that the fibrofatty residuum of aninvoluted IH represents “normal” tissue. To the contrary, many of themethylation abnormalities discovered by this study are either stable orprogressive from early to late clinical stages. For instance, the H19promoter is significantly demethylated in all IH samples (see FIG.9A-9C). However, the demethylation is progressive with age (S7Supplementary Information.) Furthermore, it was also found focaldemethylation at Exon 9 and hypermethylation at DMR0, deviating from theexpected 50% for these known imprinted sites. Both findings remainedconsistent in all IH clinical types and were age independent (S8 and S9Supplementary Information for details.) If IH tissues were beingreplaced by normal fat it would be expect the methylation abnormalitiesdemonstrated in this work to normalize, not remain constant or evenprogress with age. Given this argument, the simplest explanation for themethylation data is that IH tissue begins as epigenetically abnormalvascular stroma and transforms into epigenetically abnormal fat or is atleast replaced by the like.

CTCF BS6 Genotypes Correlate with Clinical Outcomes

Mechanisms aside, parent of origin specific effects are demonstrated atthe molecular level regarding expression patterns of both IGF2 and H19.However, the question remains whether these molecular phenotypes maytranslate into clinically significant growth patterns. FIG. 12 shows acomplete table of all patients participating in this retrospectiveclinical study. For details of subject inclusion please see methodssection. Size of the lesion as well as the date of examination wasincluded with relevant clinical information such as sex, medicaltreatments utilized and presence of ulceration during clinical course.Each patient was sorted according to CTCF Binding Site Six Genotype andpaternal contribution for heterozygotes. By plotting the size of IHlesions against the CTCF BS6 genotypes, four distinct growth curvesemerge (FIGS. 10A and 10B).

See FIG. 12: Summary of clinical data. Retrospectively collected resultswith associated descriptive information.

As shown in FIG. 12, many of the patients were treated with a corticalsteroid-methyl pregnisone, either by injection or systemic oral intake.Table 1 shows that the patients with a non-TT allelic expression had amuch better response to this medication, wherein only 25% had a failureof response to the medication. In contrast, the patients with as TTallelic expression had the least effective response to the Corticalsteroid treatment.

TABLE 1 Allelic Expression Failure of Steroids Non-Failure of SteroidsTT 7 2 Non-TT 5 15

The results of this analysis shows that the type of allelic expressioncan be used to predict the effectiveness of Cortical steroid treatment.The odds ratio from this stud is 10.5 that the TT group will failsteroids, with a sensitivity of 58.33% (95% CI 27-84%) and a specificityof 88.33% (95% CI 63-98%). The failure of steroids is defined in thisanalysis as the patient requiring surgical treatment despite thetreatment with the medication, cortical steroids.

FIG. 10. Clinical Correlation of Hernangioma Growth Rates with ParentalContributions to CTCF BS6. FIG. 10A: This retrospective analysis of 29samples, 9 TT, 20 non TT, demonstrates significantly distinct growthcurves over a large age range. The ANCOVA model has identified age as apredictor of size p=0.007 The association between tumor size and age issignificantly different among the genotypes of TT, C/T, T/C and CCp<0.0001. Of Note the paternal contribution is presented first and thematernal is second. The interaction terms of parentally specificgenotypes allowed us to test if the slopes of the curves between tumorsize and age are different among the genotypes. This analysis indictedthat an increase in 1 day of age is associated with 0.016 cm of growthin the TT group. This is significantly higher than the non TT groupp=0.019. FIG. 10B: Growth analysis focusing on the “non TT” group. Eachnon TT growth curve varied independently and significantly from the TTsamples (CC vs. TT: P<0.0001, CT vs. TT: P<0.0008. TC vs. TT: P=0.0025).Furthermore, these data suggest parent of origin specific effects asthose samples with identical genotypes but opposite parentalcontributions displayed statistically significant differences in growthcurves. The paternal T/maternal C genotype grew at approximately twicethe rate as their paternal C/maternal T carrying counterparts (p=0.5).The homozygous C group appeared to have a roughly flat growth ratebetween the heterozygotes and did not significantly vary with eitherheterozygote group (CC vs. C/T p=0.99,CC vs. T/C p=0.74)

The association between tumor size and age (days) are significantlydifferent among these four genotypes (separating heterozygotes by theirrespective parental contributions) CC, C/T, T/C, TT (p=0.0162, ACOVA.)The most impressive growth phenotype was exhibited by homozygous Tsamples reaching an average of 7.8 cm before excision (FIG. 11A)Comparing the TT group against all non-TT subjects, the difference inlesion size, increased significantly with age (p=0.0001, ANCOVA). Thusin this study, TT lesions grew more rapidly than non-TT genotypes. Infact, each growth curve—separated by maternal and paternalgenotype—varied independently and significantly from the TT samples (CCvs. TT: P<0.0001, CT vs. TT: P 0.0008, TC vs. TT: P=0.0025). Althoughgenotype appears to have no effect at approximately 100 days, afterthree months, lesions begin to distinguish themselves suggestingdistinct growth velocities. Furthermore, these data suggest parent oforigin specific effects as those samples with identical genotypes butopposite parental contributions displayed statistically significantdifferences in growth curves. Namely, those lesions carrying thepaternal T/maternal C genotype grew at approximately twice the rate astheir paternal C/maternal T carrying counterparts p=0.05 (FIG. 10B)Furthermore, each heterozygote growth curve varied by age with highcorrelation of r squared above 0.85. However, it must be emphasized thatsample size is relatively low (particularly in the C paternal/T maternalgroup) and the p-value just reached the threshold of significance.

TABLE 2 CLINICAL TABLE OF ULCERATION BY TT AND NON-TT GENOTYPE UlceratedNot Ulcerated TT 6 3 Non TT 0 20 Sensitivity = 100% Specificity = 89.96%Positive Predictive Value = 66.67% Negative Predicitve Value = 100% OddsRatio = 76.1 95% Cl = 3.4-1676 P = 0.006Retrospective collected results with associated descriptive statistics:TT Lesions have a significantly higher associated odds ratio forulceration. This proposed clinical test may be most useful in ruling outthe chance for ulceration early in the disease course as sensitivity andnegative predictive values are high. A larger prospective study iswarranted.

TT Lesions Have a Significantly Higher Associated Odds Ratio forUlceration. This proposed clinical test could be most useful in rulingout the chance of ulceration early in the disease course as sensitivityand negative predictive value are high. A larger prospective study iswarranted.

Lastly, size is a highly significant clinical outcome when studying IH.However, of similar importance is ulceration. Once an IH ulcerates, itis usually painful for the patient and is given to bleeding which can beclinically significant. Ulceration is usually a marker of rapid diseaseprogression and heralds an escalation of care. This can entail theinstitution of laser therapy, pharmacologic intervention or surgicalexcision. Not surprisingly, ulceration alone can prompt surgicaltreatment regardless of size or location of the lesion. To study therisk of ulceration an odds ratio calculation was performed comparing TTand non-TT lesions. The TT lesions had an odds ratio of 76.1 forulceration p=0.06 (Table 2). Although this is a small sample cohortpreliminary specificity was performed, sensitivity and positive andnegative predictive value calculations (Table 2). These early resultssuggest the highest clinical usefulness of the proposed test in rulingout potential future ulceration. Although encouraging, these resultswill need to be corroborated prospectively in a larger cohort.

It will be apparent to those skilled in the art that variousmodifications, combinations and variations can be made in the presentinvention without departing from the spirit or scope of the invention.Specific embodiments, features and elements described herein may bemodified, and/or combined in any suitable manner. Thus, it is intendedthat the present invention cover the modifications, combinations andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

The following references are hereby incorporated by reference in theirentirety.

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What is claimed is: 1-53. (canceled)
 54. A method of risk stratificationand treatment of a tumor that expresses Boris in a patient, the methodcomprising: a) detecting the genotype of a binding site thymine/cytosinesingle nucleotide polymorphism in a sample from the patient; wherein thebinding site is within 500 base pairs of rs10732516; and b) if thepatient is heterozygous with one thymine allele and one cytosine allele,determining which allele is the maternal allele and which allele is thepaternal allele; wherein: i) when the detected genotype of thers10732516 polymorphism is TT, the patient is classified as a highestrisk patient, and the tumor is treated by surgical excision surgicalexcision or chemotherapy; or ii) when the detected genotype of the ofthe rs10732516 polymorphism is TC, where the C allele is determined tobe the maternal allele, the patient is classified as a high riskpatient, and the tumor is treated by surgical excision surgical excisionor chemotherapy.
 55. The method of claim 54, wherein the binding site iswithin 356 base pairs of rs10732516.
 56. The method of claim 54, whereinthe binding site is within 150 base pairs of rs10732516.
 57. The methodof claim 54, wherein the binding site is between and includingrs534219523 and rs2107425.
 58. The method of claim 4, wherein thebinding site is between and including rs10732516 and rs2107425.
 59. Themethod of clair wherein the binding site is between and includingrs10732516 and rs534219523.
 60. The method of claim 54, whereindetermining which allele is the maternal allele and which allele is thepaternal allele in a heterozygous genotype at the binding site, with onethymine allele and one cytosine allele, comprises determining whetherthe cytosine is methylated; whereby: when the cytosine is not methylatedthe C allele is determined to be a maternal allele; and when thecytosine is methylated the C allele is determined to be a paternalallele.
 61. The method of claim 60, wherein determining whether thecytosine is methylated comprises spectroscopy of the patients DNA. 62.The method of claim 60, wherein determining whether the cytosine ismethylated comprises bisulfite conversion and quantitative methylationsensitive pyrosequencing.
 63. The method of claim 60, whereindetermining whether the cytosine is methylated comprises directlysequencing parental DNA.
 64. The method of claim 54 wherein the tumor isbreast cancer.
 65. The method of claim 54 wherein the tumor is ovariancancer.
 66. The method of claim 54 wherein the tumor is testicularcancer.
 67. The method of claim 54 wherein the tumor is liver cancer.68. The method of claim 54 wherein the tumor is lung cancer.
 69. Themethod of claim 54 wherein the tumor is brain cancer.
 70. The method ofclaim 54 wherein the tumor is a melanoma.